During the period covered by this merit award we have continued the research project as planned in the competitive renewal application. Our specific aims (SA?) were three, each with three subprojects. The first subprojects were generally descriptive, the second generally mechanistic and the third represented the applications for cancer prognosis of the experiments from the previous subprojects. Specifically, by order of priorities, we will test the accumulative haploid sufficiency model for gene inactivation in tumors of the MMP by several approaches, including isolation of single cell clones with mutations in some of the candidate genes for haploid sufficiency, using the same strategy used for the demonstration of the functionality of Bax and MSH6 mutations. This will be complemented with another approach using crosses of knockout mice heterozygous in different genes. We will also test the hypothesis of a methylator phenotype as the preceding causative event for the manifestation of the mutator phenotype, by the unbiased analysis if genome-wide DNA methylation alteration using MS-AFLP DNA fingerprinting. We will also continue the search for novel mutator genes responsible for some of the HNPCC and sporadic cancers of the MMP, and the screening and functional analysis of MSH5. The translational aspects of the research (prognostic applications) will be continued in an accumulative manner with validation purposes. AIMS In specific aim 1 we proposed to test the hypothesis that tumors with microsatellite instability (MSI) represent a distinct molecular pathway for colon cancer, and to investigate the mechanisms underlying this microsatellite mutator phenotype (MMP) tumorigenic pathway. In specific aim 2 we proposed to test the hypothesis that cancer of the MMP unfolds gradually by the mutational inactivation of multiple genes involved in genome integrity, including different members of the mismatch repair (MMR) gene family, and to determine the functional consequences. In specific aim 3 we proposed to test the hypothesis that the escape from apoptosis represented a critical event in tumorigenesis of the MMP pathway and to analyze the underlying mechanisms.